AbstractKavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine, Kyiv The goal of publication was the analysis of cluster methods and possibility of their application in biotechnology. The evidences found in scientific literature were summarized and analyzed. This article gives a brief description of cluster analysis — basic principles, some examples of their application are given for biotechnological problems. Results of the biotechnological studies that required application of cluster methods in combination with other mathematical approaches are considered. The conclusion contains an evaluation of the performed analysis as well as recommendations on the application of cluster analysis methods in biotechnology.

The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag) on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins.

Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

AbstractThe purpose of the work was to reproduce the sterlet of the Danube and Dnieper populations using cryopreserved sperm frozen in modified cryoprotective medium. Low-temperature cryopreservation of sterlet male reproductive cells was carried out in accordance with conventional procedures of cryobiology.

As a result of the performed work, it has been found that the applied cryoprotective environment, which includes methanol, enhances the cryoprotective effect and is technologically optimal for the process of the Danube and Dnieper populations of sterlet sperm storage and reproduction. Fish sperm cells retained viability after defrosting; their ability to fertilize the eggs was restored. The results of the conducted studies indicate that, despite a certain decrease in the quality properties of sperm after freezing/defrosting, its ability to fertilize did not significantly decrease. Revived in this way larvae from two populations of sterlet were used to reproduce repairing brood stock under conditions of fish farming.

Such a method of sterlet sperm cryopreservation could be recommended for use at commercial enterprises with sturgeon fish commodity cultivation.

АбстрактThe aim of the research was to investigate the accumulation of biobutanol by strains C. acetobutylicum using alternative substrates (Jerusalem artichoke juice, technical glycerol, crude glycerol, shredded biomass of soy, rape, wheat and switchgrass). In order to increase the accumulation of butanol in the process of cultivation, the cells of C.acetobutylicum were statically immobilized on carriers (belting strips, ferrite rings and Raschig rings) by the method of adsorption immobilization. The cells were precipitated by centrifugation, the supernatant was distilled and then fermentation products were determined. Gas chromatography was used to determine the presence of solvents in the culture fluid. The biggest accumulation of butanol (2 g/dm3) was at concentration of crude glycerol 16 g/dm3 in the medium, and complete inhibition of culture development ‒ at concentration of 25 g/dm3. The accumulation of butanol by the strain Clostridium acetobutylicum IMB B-7407 using fill and draw method depended on the amount of sequestered and infused medium. Immobilization of the culture using the Raschig rings allowed increasing the bioconversion to butanol twice. So it is shown the possibility to use non-traditional substrates for the production of biobutanol. Immobilization of C.acetobutylicum culture cells on carriers increased the accumulation of butanol. The use of Raschig rings, as carriers for immobilization, allowed increasing the accumulation of butanol twice.

AbstractThe aim of the research was to investigate the ability of new complexes as specific single-stranded oligonucleotides ‒ ODN ‒ with dimethylaminomethylmethacrylate ‒ DMAEM ‒ based polymer carriers to inhibit the expression of the physiological prion. It has been established that the introduction of complexes of newly synthesized carriers based on dimethylaminomethyl methacrylate - PEG-DMAEM-MP-27 (magnetic particles) (MP-27), PEG- DMAEM-MP-2 (MP-2), PEG- DMAEM-MP-3 (MP-3) with as ODH into the organism of rats leads to a decrease in the physiological prion content in the tissues of the spleen and small intestine. The influence of complexes of newly synthesized carriers MP-27, MP-2 and MP-3 with as specific single-stranded oligonucleotides ODH on hematological and biochemical blood parameters of Wistar rats was also studied.

The state of water in the hydrated sucrose powder, in its melt, and composite systems containing 3 parts of silica (A-300 or AM1) and 1 part of sucrose in air, low-polar organic solvent-deuterochloroform and CDCl3 medium with addition of strong acids has been studied by low-temperature 1H NMR spectroscopy. It was demonstrated that at the joint mechanoactivation of sucrose with nano-silica, its amorphization takes place. In this case, the adsorption of water in such a composite is not accompanied by the dissolution process in interfacial water of the sucrose immobilized on the SiO2 surface. It was found that the effect of sucrose solubility increasing accompanies the placement of the composite in a medium that does not dissolve water and sucrose, an organic solvent - deuterochloroform. It was suggested that it is due to an increase the contact area of adsorbed water clusters with the sucrose surface. The maximal concentration of sucrose, which dissolves in interfacial water, is about 30% by weight. The replacement of hydrophilic silica (A-300) by hydrophobic silica (AM1), while maintaining the concentration ratio of components and the amount of adsorbed water, reduces the free energy of water binding in the composite system. The obtained regularities can be used in the design of a new generation of enterosorbents.

The aim of this work was to study the effect of cerium dioxide nanoparticles on the germination of Scots pine (Pinus sylvestris) seeds and the subsequent physiological changes in plastid pigments and phenolic compounds accumulation in the seedlings tissues. Brief soaking of pine seeds in solutions of cerium dioxide nanoparticles (2-4 nm) increases the germinating power and seed germination by 1.3 times. In pine seedlings, germinated from six month old seeds, reduction in chlorophyll b, phenols and phenylalanine content is observed in 14 days after treatment. Simultaneously, concentration of carotenoids and flavonoids increased. This indicates the overall decrease in phenylpropanoid synthesis. Hence, it is reasonable to assume that cerium dioxide nanoparticles, having entered the cells of Scots pine seeds, are engaged in the regulation of phenol and terpenoid synthesis in seedlings, which greatly affects the intensity of their growth and development.

The aim of the work was to analyze the method of artificial neural networks and to examine its implementation in biotechnology. Nearly 300 publications are reviewed because this method is very widely used. The artificial neural networks are described and analyzed, and the examples of their application in biology and medicine are given. Solutions of complex problems, which required combining this method with other modern mathematical methods, are examined. Recommendations are presented for the application of this method in biotechnology.

The aim of the research was to compare the characteristics of nanoparticles with different chemical structure and size (colloidal gold Gold 1 and Gold 2, calcium phosphate CaP and poly(lactideco-glykolid) PLGA 1 and 2) to find the most efficient carriers of antigen — recombinant diphtheria toxoid for per os immunization. According to the MTT test, all studied particles show no significant cytotoxic impact on the studied cells in vitro, with the exception of CaP nanoparticles, which in high concentrations have cytotoxic effect on the U937 cells, and Gold nanoparticles 1 and 2, that are able to inhibit growth of the L929 cells. The most effective phagocytosis by macrophage-like cells J774 is observed for PLGA nanoparticles 1 and 2 with the immobilized antigen, while Gold nanoparticles 1 and 2 with antigen can interact with the surface of these cells without being phagocytated by them. In BALB/c mice immunized per os with antigen immobilized on PLGA 1 and 2 as well as Gold 2 carriers, the concentration of specific IgA antibodies in blood significantly increases after the second immunization, compared with controls. In the group of mice treated with PLGA 2 conjugated antigen, the concentration of specific IgG in blood after the third immunization also increases. These results show the promise of nanoparticles PLGA 1 and 2 as adjuvant for immunization per os.

The purpose of the research was to establish Acinetobacter sp. IMB B-7005 cultivation conditions, which provide the maximal synthesis of microbial exopolysaccharide ethapolan on a mixture of molasses and sunflower oil, and to explore the possibility of replacing refined oil in a mixture with molasses for waste one. On the basis of theoretical calculations of energy consumption for the synthesis of ethapolan and biomass, it was determined that the optimal molar ratio of the concentrations of energy-deficient (sucrose) and energy-excessive (sunflower oil) substrates in the mixture was 1.0:0.9. Experiments have shown that the highest values of exopolysaccharide synthesis were observed at a molar ratio of monosubstrates in mixture 1.0:1.1, which is as close as possible to the theoretically calculated one. It was shown that increasing concentration of molasses and refined oil in mixture from 1.0 to 1.5% was accompanied by increase in amount of synthesized exopolysaccharide and its synthesizing capacity by 1.2 and 1.3 times, respectively. The possibility of replacing refined oil in a mixture with molasses for various types of waste (after frying potatoes, meat, vegetables and mixed) was established. The maximum parameters of exopolysaccharide synthesis (concentration 14 g/l, synthesizing capacity 3.5 g exopolysaccharide/g biomass) were observed when using mixed waste oil for both inoculum obtaining and EPS biosynthesis. The obtained results testify to the possibility of development of universal technology for obtaining microbal exopolysaccharide ethapolan on a mixture of waste (molasses and waste oil) independent of the type and provider of waste oil.

The aim of this research was to study the effect of glutamine deprivation on the expression of genes encoding for HTRA1/PRSS11, LONP1/PRSS15, and some cathepsins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1). It was shown that in control glioma cells (transfected by empty vector) glutamine deprivation up-regulated the expression of LONP1, CTSD, CTSF, CTSO, and CTSS genes, down-regulated HTRA1, CTSC, and CTSK gene expressions, and did not significantly change the expression of CTSA, CTSB, and CTSL genes. Inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified the effect of glutamine deprivation on the expression of HTRA1, LONP1, CTSD, CTSL, CTSO, and CTSS genes: removed the effect of glutamine deprivation on HTRA1 and CTSO genes, introduces on CTSL gene, reduced — on CTSD gene, and enhanced — on LONP1 and CTSS genes. Therefore, glutamine deprivation affect the expression level of most studied genes in relation to the functional activity of IRE1 enzyme, a central mediator of endoplasmic reticulum stress, which responsible for control of cell proliferation and tumor growth.

The aim of the work was to optimize the process of germanium bioleaching from the dumps after coal beneficiation, namely, to determine the optimal composition of the new nutrient medium for acidophilic chemolithotrophic bacteria ensuring the maximum recovery of valuable metal in minimum time. We optimized the method of mathematical planning adapted to the plan in Greek-Latin squares. The calculations in this approach are based on the analysis of variance. The formal design of experiments has been carried out with four operating factors at four levels. The calculations were performed in Excel. The significance of the factor levels were analyzed using the Duncan’s multiple range test, the uniformity of the variances was examined the Cochran test, and the significance of the factors was tested by the Fisher criterion for each day of the experiment. The obtained results were interpreted mathematically and biologically. The following combination of factors and their levels was recommended as optimum nutrient medium, g/dm3: KH2PO4 — 1.0; (NH4)2SO4 — 2.0; KCl — 0.1; MgSO4 — 0.5; NH4Cl — 0.5; Na2S2O3 — 5.0. The proposed composition allows the more than 90% quick extraction of germanium into the solution (in four days), which was previously impossible.

The aim of the study was to create an algorithm for controlling subclinical forms of mastitis of cows on the basis of determining the activity of lactate dehydrogenase and the number of somatic cells in milk. Milk samples were taken from conditionally positive cows according to the results of the California test; the activity of lactate dehydrogenase was determined and compared with the content of somatic cells in milk.

According to the results of the analyzes, 2 out of 20 milk samples had low values of lactate dehydrogenase activity, an increased number of somatic cells (more than 250 000 in 1 ml) and negative results of bacteriological examination, which may indicate on the absence of intra-infection and a physiological increase in the number of secreted somatic cells. With increased lactate dehydrogenase activity and a somatic cell level of no more than 250 000 in 1 ml, Streptococcus agalactiae or Staphylococcus aureus bacteria were isolated, indicating on a mono-infection. At the level of somatic cells from 250 000 to 500 000 in 1 ml (4 of 20 milk samples) bacteria Streptococcus agalactiae and Staphylococcus aureus were isolated, indicative on of mix infections.

Thus, the determination of lactate dehydrogenase activity makes it possible to more accurately determine the presence of inflammatory processes in the udder, since the number of somatic cells can also increase with physiological changes (e. g., stress, etc.). The results obtained can be used to determine the subclinical forms of mastitis in the infected herd. Recommendations developed on the basis of this study were implemented in practice in the economy of the Chernihiv region.

SYNTHESIS OF AROMA COMPOUNDS BY Pleurotus ostreatus (Jacq.:Fr.) Kumm. CULTURED ON VARIOUS SUBSTRATES E. N. Vlasenko, J. V. Stepnevskaya, O. V. KuznetsovaThe aim of the study was to determine the intensity of synthesis of volatile aroma compounds by Pleurotus ostreatus (oyster mushroom) on sunflower husks and barley straw using sensory profile analysis and UV spectroscopy. The main cultural and morphological characteristics of the mycelial growth and development of fruiting bodies are determined: the period of mycelial development on the substrate, the time of primordial formation, the number of mushroom bunches per unit volume of substrate, the morphology of carpophores. Characteristic attributes of the aroma of dried fruiting bodies (mushroom, woody, sweet, herbaceous, fish, meat, floral, earthy, acidic, putrescent) are established and their aroma profiles are built. Sensory profile analysis of flavor of dried samples showed that the mushroom flavor of fungi cultivated on the sunflower husk is more pronounced than of those grown on barley straw. The light absorption maxima are recorded in the ranges 204–210 and 250–290 nm according to UV absorption spectra. Optimal conditions for extracting aromatics from dried fungi samples are the extraction time of 20–35 min at the boiling point of the solvent. Analysis of the UV spectra of fungal alcohol and hexane extracts showed that the intensity of the synthesis of volatile compounds is higher for strains cultivated on sunflower husks than for samples obtained on barley straw.

Abstract: The purpose of the research was to analyze current data concerning the problem of extending the life of multicellular animals and humans. The modern views about the processes of aging and prolongation of life are presented. The analysis focused on the genetic mechanisms of aging and mainly biotechnological approaches (genetic engineering, gene therapy, the use of stem cells, and the reprogramming of the genome) to prolong the life of multicellular organisms. For comparison, some traditional methods of prolonging life are described (drug therapy, exercise training, calorically restricted nutrition). This analysis allows to postulate the perspectives and advantages of using biotechnological methods for prolonging life in comparison with traditional ones.

Abstract:The aim of the work is to realize an alternative processing of toxic industrial waste into surfactants by strains Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 for remediation of environment.The studied strains were grown in liquid media containing such sources of carbon as waste (fried) sunflower oil, technical glycerol (by-product of biodiesel production), and aromatic compounds. The synthesis of surfactants was evaluated by emulsification index, conditional concentration of surfactants and concentration of extracellular surfactants, which was determined gravimetrically after their extraction from supernatant by the mixture of methanol and chloroform. The concentration of oil in water and soil was analyzed by gravimetric method after extraction with hexane.It was shown that with increasing concentration of the inoculum up to 10−15% and two times increase of nitrogen source content in medium containing 7−8% (v/v) of crude glycerol, concentration of surfactants synthesized by R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B 7241 and N. vaccinii IMV B-7405 was 3.4; 5.0 and 5.3 g/l, respectively, that is 1.6−1.7 times higher as compared with values on basal medium with the same content of substrate. The maximum concentration (3.9−4.3 g/l) of surfactants synthesized by A. calcoaceticus IMV B-7241 on fried sunflower oil (4%) was achieved by using the inoculum grown on refined oil. The ability of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 to decompose aromatic compounds (phenol, naphthalene, toluene, hexachlorobenzene, benzoic and N-phenylanthranilic acid) with simultaneous synthesis of extracellular metabolites with surface-active and emulsifying properties was established. In the presence of surfactants in the form of culture liquid (5−10%), the degree of degradation of complex oil with heavy metal (Cu2+, Cd2+, Pb2+, 0.01−0.5 mmol) pollution in water (3−6 g/l ) and soil (20 g/kg) after 20 days was 82−92%.Bioconversion of industrial waste into surfactants for environmental technology allows recycling toxic waste, reducing costs of microbial surfactants and provides double effect of environmental purification, which is achieved in the production and use of microbial surfactants.

Abstract:The aim of the study was to investigate the ability of lipopolysaccharides of two strains of Pseudomonas chlororaphis subsp. aureofaciens to inhibit in vitro the reproduction of human viruses: influenza A/FM/1/47 (H1N1), herpes simplex type 2 and bovine diarrhea, which is used as a model of hepatitis C virus, as well as to suppress hepatitis C virus production in model system of cells transfected with cDNA of this virus. It has been established that for both lipopolysaccharides in three types of cultures (MDCK, Vero and MDBK) the toxicity is not manifested even in a concentration of 100.0 μg/ml, and decreasing in infectious virus titer more than by 2.0 lg TCD50 (ED99) was already achieved at concentrations of 1.55 mg/ml. Selectivity indexes determination of lipopolysaccharides preparations against the influenza A/FM/1/47 (H1N1) virus, herpes simplex virus type 2 and bovine diarrhea virus shows that lipopolysaccharides of P. chlororaphis subsp. aureofaciens UCM B-306 and UCM B-111 are effective inhibitors of investigated viruses reproduction: selectivity index is at least 64. In the model of Jurkat cells transfected with human hepatitis C virus cDNA, viral RNA loading was determined in cells treated with lipopolysaccharides of P. chlororaphis subsp. aureofaciens. The results of the studies indicate that when lipopolysaccharides of both strains are administered, the production of the hepatitis C virus is completely inhibited.

Abstract:The aim of the study was to identify the influence of hydrodensified nanosilica particles on the binding of water by milled plant raw materials in neutral and acidic media. Flowers of Hibiscus sabdariffa and Calendula officinalis were used as the model materials. According to the microphotographs and low temperature 1H NMR spectroscopy data, the silica film forms on the surface of the milled plant particles, and it can significantly influence their hydration. According to the suggested scheme, some of the water from the inner cavities of plant raw materials moves (as evidenced by the decreasing radius of water-filled pores) to the zone of contact of the composite components (the radius of clusters of adsorbed water increases). In studies of desorption of active substances from milled medicinal herbs and their composites by the initial and hydrodensified nanosilica, it has been shown that the formation of a composite significantly reduces the rate of desorption. Minimal desorption is observed in composites containing hydrodensified nanosilica. The studied composite systems are promising for biomedical researches.

Abstract:The aim of the research was to select and optimize marker systems for identification of Psy1 genes alleles, which are responsible for different levels of carotenoid pigments accumulation in wheat grains, as well as to screen varieties for the selection of valuable genotypes. 162 wheat samples were analyzed by the polymerase chain reaction method. Among them, varieties and lines with different allelic states of Psy-A1 and Psy-B1 genes were identified. The Psy-D1 gene did not show any polymorphism. As a result, samples with valuable alleles of Psy1 genes, which potentially contain increased carotenoids content in grains, were selected.

Abstract: The aim of investigation was to develop a new approach in Cd, Zn, Cu, Pb phytotoxicity assessing. This approach provided the probit analysis using. Methods included probit analysis, thin layer chromatography, statistical methods (calculation of the least significant difference, correlation analysis). We applied «doze-effect» model to obtain the ranking of the metals according to their phytotoxicity in spring barley field. We offered to estimate the phytotoxicity by PhLD50 index. Research results were: graphic formalization of "dose-effect" dependence and calculation of phytotoxic doses (PhLD50 and PhLD95) for Cd, Cu, Pb, Zn in polluted soil. According to PhLD50 value we conducted a comparative assessment of Cd, Cu, Pb, Zn phytotoxicity relatively to spring barley. According to PhLD50 value metals could be ranked: Cd> Cu> Pb> Zn. The most toxic metal was Cd. PhLD values, on which the estimation of metals phytotoxicity, were: Cd – 50, Cu –129, Pb –537, Zn –603 mg / kg mobile forms in turf-podzol sandy loam soil. Our findings are relevant to estimating the metal hazard and controlling the condition of the crop growth.